Davies J, Jiang L, Pan LZ, LaBarre MJ, Anderson D, Reff M. ESKM, there was no difference in half-life or biodistribution in HLA-A(RMF/A2) (12). WT1 is an important, immunologically validated oncogenic target that has been the focus of many vaccine trials (13). WT1 is a zinc finger transcription factor with limited expression in normal adult tissues, but is over expressed in the majority of leukemias and a WF 11899A wide range of solid tumors, especially mesothelioma and ovarian cancer (14C16). WT1 was ranked as the top cancer antigenic target for immunotherapy by a National Institutes WF 11899A of Health-convened panel (17); further, WT1 expression is a biomarker and a prognostic indicator in leukemia (18, 19). ESK1 mAb specifically bound to leukemias and solid tumor cell lines that are both WT1+ and HLA-Aagainst several WT1+ HLA-A(kindly provided by Vladimir Ponomarev, MSKCC). Luciferase+/GFP+ leukemia was then expanded in NSG mice, luciferase signal was confirmed by bioluminescent imaging, and tumor cells were harvested and sorted for CD45. Peptides for T2 pulsing assays were purchased and synthesized by Genemed Synthesis, Inc. Peptides were > 90% pure. GFP+ luciferase-expressing SET2 and JMN cells were generated as described previously (12). All cells were HLA typed by the Department of Cellular Immunology at Memorial Sloan-Kettering Cancer Center. Animals C57BL/6 and C57BL/-Tg (HLA-A2.1) 1 Enge/J (6C8 week-old male), and NOD.Cg-(6C8 week-old male), known as CB17 SCID, were purchased from Taconic. All studies were conducted in accordance with IACUC approved protocols. Antibody-dependent cellular cytotoxicity (ADCC) After informed consent on Memorial Sloan-Kettering Cancer Center Institutional Review Board (MSKCC IRB) approved protocols, peripheral blood mononuclear cells WF 11899A (PBMCs) from healthy donors were obtained by Ficoll density centrifugation. Target cells used for ADCC were T2 cells pulsed with or without WT1 or RHAMM-3 peptides, and cancer cell lines or primary ovarian cancer sample without peptide pulsing. ESK1, ESKM or isotype control human IgG1 (Eureka Therapeutics, Inc) at various concentrations were incubated with target cells and fresh PBMCs at different effector: target (E:T) ratio. Cytotoxicity was measured by standard 4 hour 51Cr-release assay. Therapy of WF 11899A ESK1 and ESKM in human mesothelioma, AML and ALL xenograft mouse models Luciferase-expressing JMN cells (3105) were injected into the intraperitoneal cavity of CB17 SCID mice. On day 4, tumor engraftment was confirmed by luciferase imaging, signal was quantified with Living Image software (Xenogen), and mice were sorted into groups with similar average signal from the supine position. Mice were injected intraperitoneally with 50g ESK1, ESKM or human isotype IgG1 antibody twice weekly beginning on day 4. For AML leukemia studies, luciferase-expressing SET2 (AML) cells (3106) were injected intravenously via tail vein into NSG mice. Animals were sorted, and, where indicated, treated with intraperitoneal injections of 100g ESKM twice weekly beginning on day 6. For ALL studies, fresh leukemia cells were obtained as describe above (Cell lines and reagents) then injected intravenously into NSG mice (55106/animal), and engraftment was confirmed by bioluminescent imaging on day 2 post-injection. Animals were sorted into two groups (n=5 each) so that average signal in each group was equal. ESKM or isotype control antibody (100g/animal) was administered via retro-orbital injection on days 2, 5, 9, 12, 14 and 23, and leukemia growth was followed by bioluminescent imaging. On day 41, animals were sacrificed and bone marrow cells were harvested and pooled: after dissection and homogenization, cells were centrifuged, subjected to Ficoll density centrifugation, and counted after red blood cell lysis (acetic acid). An equal number of cells from each treatment group was resuspended in matrigel (200L/injection) and engrafted subcutaneously into the opposite shoulders of NSG mice (n=4). No further treatment was given, and tumor growth was followed by bioluminescent imaging. Pharmacokinetic and biodistribution studies Antibody was labeled with 125I (PerkinElmer) using the chloramine-T method. 100g antibody was reacted with 1mCi 125I and 20g chloramine-T, quenched with 200g Na metabisulfite, then separated from free 125I using a 10DG column equilibrated with 2% bovine serum albumin in PBS. Specific activities of products were in the range of 4C8 mCi/mg. Radiolabeled mAb was injected into mice retro-orbitally, and blood and/or organs were collected at various time points, weighed and measured on a gamma counter. HVH3 Toxicity studies For isolated cell binding studies, C57BL6/J or HLA-A2.1+ transgenic mice were sacrificed, and.